Biochemical characterization of the Drosophila wingless signaling pathway based on RNA interference
Biochemical characterization of the Drosophila wingless signaling pathway based on RNA interference
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DOI：
10.1128/mcb.24.5.2012-2024.2004
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发表时间：
2004-03-01
影响因子：
5.3
通讯作者：
Yanagawa, S
中科院分区：
文献类型：
--
作者：
Matsubayashi, H;Sese, S;Yanagawa, S
通过泛素介导的降解调节Armadillo(ARM)蛋白水平在无翼(Wg)信号中起着核心作用。虽然Zust-White 3(Zw3)介导的ARM磷酸化与其降解有关，但我们最近发现酪蛋白激酶Ipha(CKIpha)也使ARM磷酸化并诱导其降解。然而，目前尚不清楚CKIpha和Zw3以及ARM降解复合体的其他成分如何调节ARM的磷酸化以响应Wg。特别是，Wg信号在体内是否抑制CKIpha或Zw3介导的手臂磷酸化尚不清楚。为了澄清这些问题，我们在果蝇S2R+细胞中进行了一系列基于RNA干扰(RNAi)的分析，方法是使用特异性识别不同丝氨酸残基上磷酸化的手臂的抗体。这些分析表明，丝氨酸-56位和苏氨酸-52位、丝氨酸-48位和丝氨酸-44位的手臂磷酸化分别由CKIpha和Zw3介导，而Zw3直接的手臂磷酸化需要CKIpha介导的启动磷酸化。大新可刺激Zw3-，但不能刺激CKIpha介导的手臂磷酸化。Wg抑制Zw3介导的ARM磷酸化，但不抑制CKIpha介导的ARM磷酸化，这表明Wg信号中一个重要的调节步骤是Zw3介导的ARM磷酸化。此外，进一步基于RNAi对Wg途径的其他方面的分析表明，Wg诱导的凌乱的磷酸化是由CKIpha引起的，并且早老素和蛋白激酶A在调节果蝇组织培养细胞的ARM蛋白水平中发挥的作用很小。
Regulation of Armadillo (Arm) protein levels through ubiquitin-mediated degradation plays a central role in the Wingless (Wg) signaling. Although zeste-white3 (Zw3)-mediated Arm phosphorylation has been implicated in its degradation, we have recently shown that casein kinase Ialpha (CKIalpha) also phosphorylates Arm and induces its degradation. However, it remains unclear how CKIalpha and Zw3, as well as other components of the Arm degradation complex, regulate Arm phosphorylation in response to Wg. In particular, whether Wg signaling suppresses CKIalpha- or Zw3-mediated Arm phosphorylaytion in vivo is unknown. To clarify these issues, we performed a series of RNA interference (RNAi)-based analyses in Drosophila S2R+ cells by using antibodies that specifically recognize Arm phosphorylated at different serine residues. These analyses revealed that Arm phosphorylation at serine-56 and at threonine-52, serine-48, and serine-44, is mediated by CKIalpha and Zw3, respectively, and that Zw3-directed Arm phosphorylation requires CKIalpha-mediated priming phosphorylation. Daxin stimulates Zw3- but not CKIalpha-mediated Arm phosphorylation. Wg suppresses Zw3- but not CKIalpha-mediated Arm phosphorylation, indicating that a vital regulatory step in Wg signaling is Zw3-mediated Arm phosphorylation. In addition, further RNAi-based analyses of the other aspects of the Wg pathway clarified that Wg-induced Dishevelled phosphoylation is due to CKIalpha and that presenilin and protein kinase A play little part in the regulation of Arm protein levels in Drosophila tissue culture cells.